Cellular Arachidonate-releasing Function of Novel Classes of Secretory Phospholipase A2s (Groups III and XII)
2003; Elsevier BV; Volume: 278; Issue: 12 Linguagem: Inglês
10.1074/jbc.m211325200
ISSN1083-351X
AutoresMakoto Murakami, Seiko Masuda, Satoko Shimbara, Sofiane Bezzine, M Lazdunski, Gérard Lambeau, Michael H. Gelb, Satoshi Matsukura, Fumio Kokubu, Mitsuru Adachi, Ichiro Kudo,
Tópico(s)Nitric Oxide and Endothelin Effects
ResumoHere we report cellular arachidonate (AA) release and prostaglandin (PG) production by novel classes of secretory phospholipase A2s (sPLA2s), groups III and XII. Human group III sPLA2 promoted spontaneous AA release, which was augmented by interleukin-1, in HEK293 transfectants. The central sPLA2 domain alone was sufficient for itsin vitro enzymatic activity and for cellular AA release at the plasma membrane, whereas either the unique N- or C-terminal domain was required for heparanoid-dependent action on cells to augment AA release, cyclooxygenase-2 induction, and PG production. Group III sPLA2 was constitutively expressed in two human cell lines, in which other sPLA2s exhibited different stimulus inducibility. Human group XII sPLA2 had a weak enzymatic activity in vitro and minimally affects cellular AA release and PG production. Cells transfected with group XII sPLA2 exhibited abnormal morphology, suggesting a unique functional aspect of this enzyme. Based on the present results as well as our current analyses on the group I/II/V/X sPLA2s, general properties of cellular actions of a full set of mammalian sPLA2s in regulating AA metabolism are discussed. Here we report cellular arachidonate (AA) release and prostaglandin (PG) production by novel classes of secretory phospholipase A2s (sPLA2s), groups III and XII. Human group III sPLA2 promoted spontaneous AA release, which was augmented by interleukin-1, in HEK293 transfectants. The central sPLA2 domain alone was sufficient for itsin vitro enzymatic activity and for cellular AA release at the plasma membrane, whereas either the unique N- or C-terminal domain was required for heparanoid-dependent action on cells to augment AA release, cyclooxygenase-2 induction, and PG production. Group III sPLA2 was constitutively expressed in two human cell lines, in which other sPLA2s exhibited different stimulus inducibility. Human group XII sPLA2 had a weak enzymatic activity in vitro and minimally affects cellular AA release and PG production. Cells transfected with group XII sPLA2 exhibited abnormal morphology, suggesting a unique functional aspect of this enzyme. Based on the present results as well as our current analyses on the group I/II/V/X sPLA2s, general properties of cellular actions of a full set of mammalian sPLA2s in regulating AA metabolism are discussed. secretory phospholipase A2 phospholipase A2 cytosolic PLA2α cyclooxygenase arachidonic acid oleic acid linoelic acid prostaglandin prostaglandin E2 phosphatidylcholine phosphatidylethanolamine heparan sulfate proteoglycan fetal calf serum nordihydroguaiaretic acid reverse transcriptase-PCR interleukin-1 tumor necrosis factor α interferon-γ phosphate-buffered saline Tris-buffered saline wild type 1-palmitoyl-2-[14C]linoleoyl-PE 1-palmitoyl-2-[14C]linoleoyl-PC 1-palmitoyl-2-[14C]arachidonoyl-PC 1-palmitoyl-2-[14C]archidonoyl-PE Secretory phospholipase A2(sPLA2)1comprises a family of Ca2+-dependent lipolytic enzymes with a conserved Ca2+-binding loop and His-Asp dyad at the catalytic site (1Murakami M. Kudo I. Adv. Immunol. 2001; 77: 163-194Crossref PubMed Scopus (134) Google Scholar, 2Valentin E. Lambeau G. Biochim. Biophys. Acta. 2000; 1488: 59-70Crossref PubMed Scopus (313) Google Scholar). To date, 10 sPLA2 enzymes (groups IB, IIA, IIC, IID, IIE, IIF, V, X, III, and XII) have been identified in mammals (1Murakami M. Kudo I. Adv. Immunol. 2001; 77: 163-194Crossref PubMed Scopus (134) Google Scholar, 2Valentin E. Lambeau G. Biochim. Biophys. Acta. 2000; 1488: 59-70Crossref PubMed Scopus (313) Google Scholar). In general, sPLA2s exhibit tissue- and species-specific expression, which suggests that their cellular behaviors and functions differ. The group I/II/V/X sPLA2s represent a class of enzymes with a molecular mass of 14–18 kDa and 6–8 conserved disulfides (1Murakami M. Kudo I. Adv. Immunol. 2001; 77: 163-194Crossref PubMed Scopus (134) Google Scholar, 2Valentin E. Lambeau G. Biochim. Biophys. Acta. 2000; 1488: 59-70Crossref PubMed Scopus (313) Google Scholar). sPLA2-IB and -X, but not the other enzymes, have an N-terminal prepropeptide, and the proteolytic cleavage of this prepropeptide is a regulatory step for generation of an active enzyme (3Tojo H. Ono T. Kuramitsu S. Kagamiyama H. Okamoto M. J. Biol. Chem. 1988; 263: 5724-5731Abstract Full Text PDF PubMed Google Scholar, 4Hanasaki K. Ono T. Saiga A. Morioka Y. Ikeda M. Kawamoto K. Higashino K. Nakano K. Yamada K. Ishizaki J. Arita H. J. Biol. Chem. 1999; 274: 34203-34211Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). sPLA2-IB is abundantly present in pancreatic juice, and its main function has been thought to be the digestion of dietary phospholipids, although recent data with sPLA2-IB knockout mice have demonstrated no appreciable defects in this process (5Richmond B.L. Boileau A.C. Zheng S. Huggins K.W. Granholm N.A. Tso P. Hui D.Y. Gastroenterology. 2001; 120: 1193-1202Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). sPLA2-IIA, a prototypic inflammatory PLA2, and other group II subfamily sPLA2s (IID, IIE, IIF, and V) are inducible in various tissues with inflammation or damage (6Pfeilschifter J. Schalkwijk C. Briner V.A. van den Bosch H. J. Clin. Invest. 1993; 92: 2516-2523Crossref PubMed Scopus (208) Google Scholar, 7Pruzanski W. Vadas P. Immunol. Today. 1991; 12: 143-146Abstract Full Text PDF PubMed Scopus (397) Google Scholar, 8Oka S. Arita H. J. Biol. Chem. 1991; 266: 9956-9960Abstract Full Text PDF PubMed Google Scholar, 9Murakami M. Yoshihara K. Shimbara S. Sawada M. Inagaki N. Nagai H. Naito M. Tsuruo T. Moon T.C. Chang H.W. Kudo I. Eur. J. Biochem. 2002; 269: 2698-2707Crossref PubMed Scopus (27) Google Scholar, 10Murakami M. Yoshihara K. Shimbara S. Lambeau G. Gelb M.H. Singer A.G. Sawada M. Inagaki N. Nagai H. Kudo I. Biochem. Biophys. Res. Commun. 2002; 292: 689-696Crossref PubMed Scopus (48) Google Scholar, 11Murakami M. Yoshihara K. Shimbara S. Lambeau G. Gelb M.H. Singer A.G. Sawada M. Inagaki N. Nagai H. Ishihara M. Ishikawa Y. Ishii T. Kudo I. J. Biol. Chem. 2002; 277: 19145-19155Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 12Sawada H. Murakami M. Enomoto A. Shimbara S. Kudo I. Eur. J. Biochem. 1999; 263: 826-835Crossref PubMed Scopus (83) Google Scholar, 13Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (170) Google Scholar). On the bases of current biochemical and cell biological studies, sPLA2s in the I/II/V/X branch may participate in various biological events, including arachidonate (AA) release from cellular membranes (see below) (9Murakami M. Yoshihara K. Shimbara S. Sawada M. Inagaki N. Nagai H. Naito M. Tsuruo T. Moon T.C. Chang H.W. Kudo I. Eur. J. Biochem. 2002; 269: 2698-2707Crossref PubMed Scopus (27) Google Scholar, 10Murakami M. Yoshihara K. Shimbara S. Lambeau G. Gelb M.H. Singer A.G. Sawada M. Inagaki N. Nagai H. Kudo I. Biochem. Biophys. Res. Commun. 2002; 292: 689-696Crossref PubMed Scopus (48) Google Scholar, 11Murakami M. Yoshihara K. Shimbara S. Lambeau G. Gelb M.H. Singer A.G. Sawada M. Inagaki N. Nagai H. Ishihara M. Ishikawa Y. Ishii T. Kudo I. J. Biol. Chem. 2002; 277: 19145-19155Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 12Sawada H. Murakami M. Enomoto A. Shimbara S. Kudo I. Eur. J. Biochem. 1999; 263: 826-835Crossref PubMed Scopus (83) Google Scholar, 13Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (170) Google Scholar, 14Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 15Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 16Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 17Murakami M. Kambe T. Shimbara S. Higashino K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 18Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), host defense against bacteria (19Weinrauch Y. Abad C. Liang N.S. Lowry S.F. Weiss J. J. Clin. Invest. 1998; 102: 633-639Crossref PubMed Scopus (128) Google Scholar, 20Koduri R.S. Gronroos J.O. Laine V.J. Le Calvez C. Lambeau G. Nevalainen T.J. Gelb M.H. J. Biol. Chem. 2002; 277: 5849-5857Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar), atherosclerosis (21Laine V.J. Grass D.S. Nevalainen T.J. J. Immunol. 1999; 162: 7402-7408PubMed Google Scholar, 22Tietge U.J. Maugeais C. Cain W. Grass D. Glick J.M. de Beer F.C. Rader D.J. J. Biol. Chem. 2000; 275: 10077-10084Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), blood coagulation (23Mounier C. Franken P.A. Verheij H.M. Bon C. Eur. J. Biochem. 1996; 237: 778-785Crossref PubMed Scopus (46) Google Scholar), and cancer (24MacPhee M. Chepenik K.P. Liddell R.A. Nelson K.K. Siracusa L.D. Buchberg A.M. Cell. 1995; 81: 957-966Abstract Full Text PDF PubMed Scopus (525) Google Scholar). Beyond the essential role of cytosolic PLA2α (cPLA2α) in the initiation of stimulus-coupled AA metabolism, the I/II/V/X class of sPLA2s also has the ability to augment AA metabolism by multiple mechanisms (1Murakami M. Kudo I. Adv. Immunol. 2001; 77: 163-194Crossref PubMed Scopus (134) Google Scholar). sPLA2s that show high interfacial binding to zwitterionic phosphatidylcholine (PC), such as sPLA2-X and -V, are capable of releasing AA from the PC-rich outer leaflet of the plasma membrane of quiescent cells (the external plasma membrane pathway) (17Murakami M. Kambe T. Shimbara S. Higashino K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 25Bezzine S. Koduri R.S. Valentin E. Murakami M. Kudo I. Ghomashchi F. Sadilek M. Lambeau G. Gelb M.H. J. Biol. Chem. 2000; 275: 3179-3191Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 26Han S.K. Kim K.P. Koduri R. Bittova L. Munoz N.M. Leff A.R. Wilton D.C. Gelb M.H. Cho W. J. Biol. Chem. 1999; 274: 11881-11888Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 27Morioka Y. Ikeda M. Saiga A. Fujii N. Ishimoto Y. Arita H. Hanasaki K. FEBS Lett. 2000; 487: 262-266Crossref PubMed Scopus (57) Google Scholar). Cationic, heparin-binding, group II subfamily sPLA2s, such as sPLA2-IIA, -IID, -IIE, and -V, show marked preference for anionic phospholipids over PC and utilize the heparan sulfate proteoglycan (HSPG)-shuttling pathway (14Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 15Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 16Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 17Murakami M. Kambe T. Shimbara S. Higashino K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 18Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 28Kim Y.J. Kim K.P. Rhee H.J. Das S. Rafter J.D. Oh Y.S. Cho W. J. Biol. Chem. 2002; 277: 9358-9365Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In this regulatory pathway, these enzymes are captured by HSPGs (typically glypican, a glycosylphosphatidylinositol-anchored HSPG) in caveolae or rafts on activated cells and then internalized into vesicular membrane compartments that are enriched in the perinuclear area, where downstream cyclooxygenases (COXs) are located (16Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 17Murakami M. Kambe T. Shimbara S. Higashino K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 28Kim Y.J. Kim K.P. Rhee H.J. Das S. Rafter J.D. Oh Y.S. Cho W. J. Biol. Chem. 2002; 277: 9358-9365Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). This spatiotemporal co-localization of sPLA2s and COXs in the perinuclear compartments may allow efficient supply of AA between these enzymes. Recent evidence implies that the clathrin-independent, caveolae/raft-mediated endocytosis, and associated vesicular traffic is directed toward a rapid cycling pathway via the Golgi and endoplasmic reticulum (29Nichols B.J. Lippincott-Schwartz J. Trends Cell Biol. 2001; 11: 406-412Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). Occurrence of the HSPG-shuttling pathway appears to be cell type- and stimulus-specific, and in certain cases HSPGs exhibit a negative regulatory effect on the heparin-binding sPLA2s by facilitating their internalization and subsequent lysosomal degradation (30Kim K.P. Rafter J.D. Bittova L. Han S.K. Snitko Y. Munoz N.M. Leff A.R. Cho W. J. Biol. Chem. 2001; 276: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 31Enomoto A. Murakami M. Kudo I. Biochem. Biophys. Res. Commun. 2000; 276: 667-672Crossref PubMed Scopus (23) Google Scholar). sPLA2-IIF, an anionic group II subfamily sPLA2with poor affinity for HSPG, may interact stably with the plasma membrane through its unique C-terminal extension and releases AA (11Murakami M. Yoshihara K. Shimbara S. Lambeau G. Gelb M.H. Singer A.G. Sawada M. Inagaki N. Nagai H. Ishihara M. Ishikawa Y. Ishii T. Kudo I. J. Biol. Chem. 2002; 277: 19145-19155Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), although the possibility that this enzyme also functions after internalization cannot be ruled out. In addition, cellular actions of several sPLA2s can be mediated by sPLA2receptors independent of their enzymatic activity (32Lambeau G. Lazdunski M. Trends Pharmacol. Sci. 1999; 20: 162-170Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Targeted disruption of the M-type sPLA2 receptor gene results in reduced inflammatory response in mice (33Hanasaki K. Yokota Y. Ishizaki J. Itoh T. Arita H. J. Biol. Chem. 1997; 272: 32792-32797Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The M-type sPLA2 receptor can also act as a negative regulator for sPLA2s by inhibiting their enzymatic functions in serum and by promoting their internalization and subsequent degradation (34Zvaritch E. Lambeau G. Lazdunski M. J. Biol. Chem. 1996; 271: 250-257Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar,35Morioka Y. Saiga A. Yokota Y. Suzuki N. Ikeda M. Ono T. Nakano K. Fujii N. Ishizaki J. Arita H. Hanasaki K. Arch. Biochem. Biophys. 2000; 381: 31-42Crossref PubMed Scopus (77) Google Scholar). Besides the I/II/V/X branch, two distinct classes of sPLA2, namely the group III and group XII branches, have been recently identified in mammals (36Valentin E. Ghomashchi F. Gelb M.H. Lazdunski M. Lambeau G. J. Biol. Chem. 2000; 275: 7492-7496Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 37Gelb M.H. Valentin E. Ghomashchi F. Lazdunski M. Lambeau G. J. Biol. Chem. 2000; 275: 39823-39826Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 38Ho I.C. Arm J.P. Bingham 3rd, C.O. Choi A. Austen K.F. Glimcher L. J. Biol. Chem. 2001; 276: 18321-18326Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Structurally, these two novel sPLA2s show homology with the I/II/V/X sPLA2s only within the Ca2+ loop and catalytic site His-Asp dyad. Human sPLA2-III is a 56-kDa protein containing a long N-terminal domain, a central sPLA2 domain that is homologous to bee venom group III sPLA2, and a long C-terminal domain (36Valentin E. Ghomashchi F. Gelb M.H. Lazdunski M. Lambeau G. J. Biol. Chem. 2000; 275: 7492-7496Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). sPLA2-XII, which harbors an unusual Ca2+ loop, is distantly related to other classes of sPLA2s (37Gelb M.H. Valentin E. Ghomashchi F. Lazdunski M. Lambeau G. J. Biol. Chem. 2000; 275: 39823-39826Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 38Ho I.C. Arm J.P. Bingham 3rd, C.O. Choi A. Austen K.F. Glimcher L. J. Biol. Chem. 2001; 276: 18321-18326Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). This enzyme is expressed in antigen-activated helper T cells in the mouse (38Ho I.C. Arm J.P. Bingham 3rd, C.O. Choi A. Austen K.F. Glimcher L. J. Biol. Chem. 2001; 276: 18321-18326Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). However, cellular functions of these two novel sPLA2s have not yet been described. To expand our current understanding of the sPLA2actions on cells, we studied the cellular AA-releasing and prostaglandin (PG)-biosynthetic properties of human sPLA2-III and -XII by expressing these enzymes in HEK293 cells, as we have previously done with the I/II/V/X sPLA2s (9Murakami M. Yoshihara K. Shimbara S. Sawada M. Inagaki N. Nagai H. Naito M. Tsuruo T. Moon T.C. Chang H.W. Kudo I. Eur. J. Biochem. 2002; 269: 2698-2707Crossref PubMed Scopus (27) Google Scholar, 10Murakami M. Yoshihara K. Shimbara S. Lambeau G. Gelb M.H. Singer A.G. Sawada M. Inagaki N. Nagai H. Kudo I. Biochem. Biophys. Res. Commun. 2002; 292: 689-696Crossref PubMed Scopus (48) Google Scholar, 11Murakami M. Yoshihara K. Shimbara S. Lambeau G. Gelb M.H. Singer A.G. Sawada M. Inagaki N. Nagai H. Ishihara M. Ishikawa Y. Ishii T. Kudo I. J. Biol. Chem. 2002; 277: 19145-19155Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 12Sawada H. Murakami M. Enomoto A. Shimbara S. Kudo I. Eur. J. Biochem. 1999; 263: 826-835Crossref PubMed Scopus (83) Google Scholar, 14Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 15Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 16Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 17Murakami M. Kambe T. Shimbara S. Higashino K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 18Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Human embryonic kidney 293 (HEK293) cells (Human Science Research Resources Bank), human lung epithelial BEAS-2B cells (American Type Cell Collection, Manassas, VA), and human colon adenocarcinoma HCA-7 cells (a generous gift from Dr. M. Tsujii (Osaka University) and Dr. R. DuBois (Vanderbilt University Medical Center and VA Medical Center)) were cultured in RPMI 1640 medium (Nissui Pharmaceutical Co.) containing 10% (v/v) fetal calf serum (FCS; Bioserum). The cDNAs for human sPLA2-III (36Valentin E. Ghomashchi F. Gelb M.H. Lazdunski M. Lambeau G. J. Biol. Chem. 2000; 275: 7492-7496Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), human sPLA2-XII (37Gelb M.H. Valentin E. Ghomashchi F. Lazdunski M. Lambeau G. J. Biol. Chem. 2000; 275: 39823-39826Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), human COX-1 and COX-2 (15Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar), and rat glypican-1 (16Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) were described previously. HEK293 cells stably expressing human sPLA2-V, human sPLA2-IIF, and human COX-2 were described previously (11Murakami M. Yoshihara K. Shimbara S. Lambeau G. Gelb M.H. Singer A.G. Sawada M. Inagaki N. Nagai H. Ishihara M. Ishikawa Y. Ishii T. Kudo I. J. Biol. Chem. 2002; 277: 19145-19155Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 14Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 15Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). The enzyme immunoassay kits for PGE2 and the COX-2 inhibitor NS-398 were purchased from Cayman Chemicals. The goat anti-human COX-1 and anti-human COX-2 antibodies were purchased from Santa Cruz Biosciences, Inc. (Santa Cruz, CA). A23187 was purchased from Calbiochem. Human interleukin (IL)-1β, interferon (IFN)-γ, and tumor necrosis factor α (TNF-α) were purchased from Genzyme. LipofectAMINE 2000 reagent, Opti-MEM medium, TRIzol reagent, geneticin, zeocin, and mammalian expression vectors (pCR3.1, pRc-CMV, and pcDNA3.1 series of vectors containing a neomycin- or zeocin-resistant gene) were obtained from Invitrogen. Fluorescein isothiocyanate-conjugated anti-mouse and anti-rabbit IgGs and horseradish peroxidase-conjugated anti-goat IgG were purchased from Zymed Laboratories Inc.. Mouse monoclonal anti-FLAG antibody, anti-FLAG antibody-conjugated agarose, and heparin were from Sigma. The lipoxygenase-inhibitory antioxidant nordihydroguaiaretic acid (NDGA) was purchased from BIOMOL. Heparin-Sepharose was purchased from Amersham Biosciences. Rabbit antiserum for human sPLA2-XII was prepared as described previously (39Degousee N. Ghomashchi F. Stefanski E. Singer A. Smart B.P. Borregaard N. Reithmeier R. Lindsay T.F. Lichtenberger C. Reinisch W. Lambeau G. Arm J. Tischfield J. Gelb M.H. Rubin B.B. J. Biol. Chem. 2001; 277: 5061-5073Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Establishment of HEK293 transformants was performed as described previously (12Sawada H. Murakami M. Enomoto A. Shimbara S. Kudo I. Eur. J. Biochem. 1999; 263: 826-835Crossref PubMed Scopus (83) Google Scholar, 13Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (170) Google Scholar, 14Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 15Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 16Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Briefly, 1 μg of plasmid (sPLA2 cDNAs subcloned into the pRc-CMV or pCR3.1 vector) was mixed with 2 μl of LipofectAMINE 2000 in 100 μl of Opti-MEM medium for 30 min and then added to cells that had attained 40–60% confluence in 12-well plates (Iwaki Glass) containing 0.5 ml of Opti-MEM. After incubation for 6 h, the medium was replaced with 1 ml of fresh culture medium. After overnight culture, the medium was replaced with 1 ml of fresh medium, and culture was continued at 37 °C in an incubator flushed with 5% CO2 in humidified air. The cells were cloned by limiting dilution in 96-well plates in culture medium supplemented with 1 mg/ml geneticin. After culture for 3–4 weeks, wells containing a single colony were chosen, and the expression of each protein was assessed by RNA blotting. The established clones were expanded and used for the experiments as described below. In order to establish double transformants expressing sPLA2and glypican, cells expressing each sPLA2 were subjected to a second transfection with glypican cDNA subcloned into pCDNA3.1/Zeo(+) using LipofectAMINE 2000. Three days after the transfection, the cells were used for the experiments or seeded into 96-well plates and cloned by culturing in the presence of 50 μg/ml zeocin to establish stable transformants. To assess functional coupling between sPLA2 and either of the two COX isozymes, cells stably expressing sPLA2 were transfected with COX-1 or COX-2 subcloned into pCDNA3.1 using LipofectAMINE 2000. Three days after the transfection, the cells were activated with A23187 to measure PGE2 generation and were subjected to immunoblotting to examine COX-1 or COX-2 expression (see below). sPLA2activity was assayed by measuring the amounts of radiolabeled fatty acids released from the substrate 1-palmitoyl-2-[14C]arachidonoyl-phosphatidylethanolamine (2-AA-PE), 1-palmitoyl-2-[14C]linoleoyl-PE (2-LA-PE), 2-AA-PC, or 2-LA-PC (Amersham Biosciences). Each substrate in ethanol was dried up under N2 stream and was dispersed in water by sonication. Each reaction mixture (total volume 250 μl) consisted of appropriate amounts of the required sample, 100 mm Tris-HCl (pH 7.4), 4 mm CaCl2, and 10 μmsubstrate. After incubation for 10–30 min at 37 °C, [14C]AA or [14C]LA was extracted, and radioactivity was quantified, as described previously (14Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 15Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 16Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 17Murakami M. Kambe T. Shimbara S. Higashino K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 18Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Baculovirus expression of recombinant sPLA2proteins was performed using the BAC-to-BAC baculovirus expression system (Invitrogen). Briefly, sPLA2 cDNAs were subcloned into the baculovirus expression vector pFASTBAC1 (Invitrogen) at appropriate restriction enzyme sites. Recombinant sPLA2proteins were first expressed in Sf9 insect cells and then amplified in High Five insect cells (Invitrogen) according to the manufacturer's instructions. Culture supernatants and cell lysates (4–5 days after infection) were used for subsequent experiments. Sf9 cells and High Five cells were maintained in Grace's insect medium (Invitrogen) supplemented with 10% FCS and Express Five SFM serum-free medium (Invitrogen), respectively. Recombinant sPLA2s (culture supernatants from baculovirus-infected High Five cells) were incubated with various amounts of heparin-Sepharose beads in 10 mmTris-HCl (pH 7.4) containing 150 mm NaCl (TBS) for 2 h at 4 °C, and PLA2 activities remaining in the supernatants were assayed. Approximately equal amounts (∼5 μg) of total RNA obtained from the cells were applied to separate lanes of 1.2% (w/v) formaldehyde-agarose gels, electrophoresed, and transferred to Immobilon-N membranes (Millipore Corp.). The resulting blots were then probed with the respective cDNA probes that had been labeled with [32P]dCTP (Amersham Biosciences) by random priming (Takara Biomedicals). All hybridizations were carried out as described previously (14Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 15Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 16Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 17Murakami M. Kambe T. Shimbara S. Higashino K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 18Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Lysates from 105 cells were subjected to SDS-PAGE using 7.5–12.5% gels under reducing conditions. The separated proteins were electroblotted onto nitrocellulose membranes (Schleicher and Schuell) using a semidry blotter (MilliBlot-SDE system; Millipore). After blocking with 3% (w/v) skim milk in TBS containing 0.05% Tween 20 (TBS-Tween), the membranes were probed with the respective antibodies (1:
Referência(s)